Nitrate transporters: an overview in legumes.

MAIN CONCLUSION: The nitrate transporters, belonging to NPF and NRT2 families, play critical roles in nitrate signaling, root growth and nodule development in legumes. Nitrate plays an essential role during plant development as nutrient and also as signal molecule, in both cases working via the activity of nitrate transporters. To date, few studies on NRT2 or NPF nitrate transporters in legumes have been reported, and most of those concern Lotus japonicus and Medicago truncatula. A molecular characterization led to the identification of 4 putative LjNRT2 and 37 putative LjNPF gene sequences in L. japonicus. In M. truncatula, the NRT2 family is composed of 3 putative members. Using the new genome annotation of M. truncatula (Mt4.0), we identified, for this review, 97 putative MtNPF sequences, including 32 new sequences relative to previous studies. Functional characterization has been published for only two MtNPF genes, encoding nitrate transporters of M. truncatula. Both transporters have a role in root system development via abscisic acid signaling: MtNPF6.8 acts as a nitrate sensor during the cell elongation of the primary root, while MtNPF1.7 contributes to the cellular organization of the root tip and nodule formation. An in silico expression study of MtNPF genes confirmed that NPF genes are expressed in nodules, as previously shown for L. japonicus, suggesting a role for the corresponding proteins in nitrate transport, or signal perception in nodules. This review summarizes our knowledge of legume nitrate transporters and discusses new roles for these proteins based on recent discoveries.

The nitrate transporter MtNPF6.8 (MtNRT1.3) transports abscisic acid and mediates nitrate regulation of primary root growth in Medicago truncatula.

Elongation of the primary root during postgermination of Medicago truncatula seedlings is a multigenic trait that is responsive to exogenous nitrate. A quantitative genetic approach suggested the involvement of the nitrate transporter MtNPF6.8 (for Medicago truncatula NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER Family6.8) in the inhibition of primary root elongation by high exogenous nitrate. In this study, the inhibitory effect of nitrate on primary root elongation, via inhibition of elongation of root cortical cells, was abolished in npf6.8 knockdown lines. Accordingly, we propose that MtNPF6.8 mediates nitrate inhibitory effects on primary root growth in M. truncatula. pMtNPF6.8:GUS promoter-reporter gene fusion in Agrobacterium rhizogenes-generated transgenic roots showed the expression of MtNPF6.8 in the pericycle region of primary roots and lateral roots, and in lateral root primordia and tips. MtNPF6.8 expression was insensitive to auxin and was stimulated by abscisic acid (ABA), which restored the inhibitory effect of nitrate in npf6.8 knockdown lines. It is then proposed that ABA acts downstream of MtNPF6.8 in this nitrate signaling pathway. Furthermore, MtNPF6.8 was shown to transport ABA in Xenopus spp. oocytes, suggesting an additional role of MtNPF6.8 in ABA root-to-shoot translocation. (15)NO3(-)-influx experiments showed that only the inducible component of the low-affinity transport system was affected in npf6.8 knockdown lines. This indicates that MtNPF6.8 is a major contributor to the inducible component of the low-affinity transport system. The short-term induction by nitrate of the expression of Nitrate Reductase1 (NR1) and NR2 (genes that encode two nitrate reductase isoforms) was greatly reduced in the npf6.8 knockdown lines, supporting a role of MtNPF6.8 in the primary nitrate response in M. truncatula.

Identification and molecular characterization of Medicago truncatula NRT2 and NAR2 families.

Nitrate transporters received little attention to legumes probably because these species are able to adapt to N starvation by developing biological N2 fixation. Still it is important to study nitrate transport systems in legumes because nitrate intervenes as a signal in regulation of nodulation probably through nitrate transporters. The aim of this work is to achieve a molecular characterization of nitrate transporter 2 (NRT2) and NAR2 (NRT3) families to allow further work that would unravel their involvement in nitrate transport and signaling. Browsing the latest version of the Medicago truncatula genome annotation (v4 version) revealed three putative NRT2 members that we have named MtNRT2.1 (Medtr4g057890.1), MtNRT2.2 (Medtr4g057865.1) and MtNRT2.3 (Medtr8g069775.1) and two putative NAR2 members we named MtNAR2.1 (Medtr4g104730.1) and MtNAR2.2 (Medtr4g104700.1). The regulation and the spatial expression profiles of MtNRT2.1, the coincidence of its expression with that of MtNAR2.1 and MtNAR2.2 and the size of the encoded protein with 12 transmembrane (TM) spanning regions strongly support the idea that MtNRT2.1 is a nitrate transporter with a major contribution to the high-affinity transport system (HATS), while a very low level of expression characterized MtNRT2.2. Unlike MtNRT2.1, MtNRT2.3 showed a lower level of expression in the root system but was expressed in the shoots and in the nodules thus suggesting an involvement of the encoded protein in nitrate transport inside the plant and/or in nitrate signaling pathways controlling post-inoculation processes that govern nodule functioning.

Abscisic acid-induced nitric oxide and proline accumulation in independent pathways under water-deficit stress during seedling establishment in Medicago truncatula.

Nitric oxide (NO) production and amino acid metabolism modulation, in particular abscisic acid (ABA)-dependent proline accumulation, are stimulated in planta by most abiotic stresses. However, the relationship between NO production and proline accumulation under abiotic stress is still poorly understood, especially in the early phases of plant development. To unravel this question, this work investigated the tight relationship between NO production and proline metabolism under water-deficit stress during seedling establishment. Endogenous nitrate reductase-dependent NO production in Medicago truncatula seedlings increased in a time-dependent manner after short-term water-deficit stress. This water-deficit-induced endogenous NO accumulation was mediated through a ABA-dependent pathway and accompanied by an inhibition of seed germination, a loss of water content, and a decrease in elongation of embryo axes. Interestingly, a treatment with a specific NO scavenger (cPTIO) alleviated these water-deficit detrimental effects. However, the content of total amino acids, in particular glutamate and proline, as well as the expression of genes encoding enzymes of synthesis and degradation of proline were not affected by cPTIO treatment under water-deficit stress. Under normal conditions, exogenous NO donor stimulated neither the expression of P5CS2 nor the proline content, as observed after PEG treatment. These results strongly suggest that the modulation of proline metabolism is independent of NO production under short-term water-deficit stress during seedling establishment.

NPF and NRT2 from Pisum sativum Potentially Involved in Nodule Functioning: Lessons from Medicago truncatula and Lotus japonicus.

In addition to absorbing nitrogen from the soil, legumes have the ability to use atmospheric N2 through symbiotic nitrogen fixation. Therefore, legumes have developed mechanisms regulating nodulation in response to the amount of nitrate in the soil; in the presence of high nitrate concentrations, nodulation is inhibited, while low nitrate concentrations stimulate nodulation and nitrogen fixation. This allows the legumes to switch from soil nitrogen acquisition to symbiotic nitrogen fixation. Recently, particular interest has been given to the nitrate transporters, such as Nitrate Transporter1/Peptide transporter Family (NPF) and Nitrate Transporter 2 (NRT2), having a role in the functioning of nodules. Nitrate transporters of the two model plants, Lotus japonicus and Medicago truncatula, shown to have a positive and/or a negative role in nodule functioning depending on nitrate concentration, are presented in this article. In particular, the following transporters were thoroughly studied: (i) members of NPF transporters family, such as LjNPF8.6 and LjNPF3.1 in L. japonicus and MtNPF1.7 and MtNPF7.6 in M. truncatula, and (ii) members of NRT2 transporters family, such as LjNRT2.4 and LjNRT2.1 in L. japonicus and MtNRT2.1 in M. truncatula. Also, by exploiting available genomic and transcriptomic data in the literature, we have identified the complete PsNPF family in Pisum sativum (69 sequences previously described and 21 new that we have annotated) and putative nitrate transporters candidate for playing a role in nodule functioning in P. sativum.

The nitrate transporter-sensor MtNPF6.8 regulates the branched chain amino acid/pantothenate metabolic pathway in barrel medic (Medicago truncatula Gaertn.) root tip.

Nitrogen is the most limiting nutrient for plants, and it is preferentially absorbed in the form of nitrate by roots, which adapt to nitrate fluctuations by remodelling their architecture. Although core mechanisms of the response to nitrate availability are relatively well-known, signalling events controlling root growth and architecture have not all been identified, in particular in Legumes. However, the developmental effect of nitrate in Legumes is critical since external nitrate not only regulates root architecture but also N2-fixing nodule development. We have previously shown that in barrel medic (Medicago truncatula), the nitrate transporter MtNPF6.8 is required for nitrate sensitivity in root tip. However, uncertainty remains as to whether nitrogen metabolism itself is involved in the MtNPF6.8-mediated response. Here, we examine the metabolic effects of MtNPF6.8-dependent nitrate signalling using metabolomics and proteomics in WT and mtnpf6.8 root tips in presence or absence of nitrate. We found a reorchestration of metabolism due to the mutation, in favour of the branched chain amino acids/pantothenate metabolic pathway, and lipid catabolism via glyoxylate. That is, the mtnpf6.8 mutation was likely associated with a specific rerouting of acetyl-CoA production (glyoxylic cycle) and utilisation (pantothenate and branched chain amino acid synthesis). In agreement with our previous findings, class III peroxidases were confirmed as the main protein class responsive to nitrate, although in an MtNPF6.8-independent fashion. Our data rather suggest the involvement of other pathways within mtnpf6.8 root tips, such as Ca2+ signalling or cell wall methylation.

The Nitrate Transporter MtNPF6.8 Is a Master Sensor of Nitrate Signal in the Primary Root Tip of Medicago truncatula.

Nitrate is not only an essential nutrient for plants, but also a signal involved in plant development. We have previously shown in the model legume Medicago truncatula, that the nitrate signal, which restricts primary root growth, is mediated by MtNPF6.8, a nitrate transporter. Nitrate signal also induces changes in reactive oxygen species accumulation in the root tip due to changes in cell wall peroxidase (PODs) activity. Thus, it was interesting to determine the importance of the role of MtNPF6.8 in the regulation of the root growth by nitrate and identify the POD isoforms responsible for the changes in POD activity. For this purpose, we compared in M. truncatula a npf6.8 mutant and nitrate insensitive line deficient in MtNPF6.8 and the corresponding wild and sensitive genotype for their transcriptomic and proteomic responses to nitrate. Interestingly, only 13 transcripts and no protein were differently accumulated in the primary root tip of the npf6.8-3 mutant line in response to nitrate. The sensitivity of the primary root tip to nitrate appeared therefore to be strongly linked to the integrity of MtNPF6.8 which acts as a master mediator of the nitrate signal involved in the control of the root system architecture. In parallel, 7,259 and 493 genes responded, respectively, at the level of transcripts or proteins in the wild type, 196 genes being identified by both their transcript and protein. By focusing on these 196 genes, a concordance of expression was observed for most of them with 143 genes being up-regulated and 51 being down-regulated at the two gene expression levels. Their ontology analysis uncovered a high enrichment in POD genes, allowing the identification of POD candidates involved in the changes in POD activity previously observed in response to nitrate.

Pisum sativum Response to Nitrate as Affected by Rhizobium leguminosarum-Derived Signals.

Legumes are suitable for the development of sustainable agroecosystems because of their ability to use atmospheric N2 through symbiotic nitrogen fixation (SNF). However, a basic NO3- input is necessary before SNF takes place to ensure successful seedling establishment. Since Rhizobia not only induce nodulation but also affect root branching by stimulating the development of lateral roots, and NO3- as a signal also modulates root system architecture, we investigated whether Rhizobium-derived signals interfere in nitrate signaling. Here, we bring evidence that (i) Rhizobium-altered NO3--mediated processes in pea expressions of major players in NO3- transport, sensing, and signaling were affected, and (ii) the characteristic limitation of root foraging and branching in response to NO3- supply was abolished. The number of tertiary roots per secondary root was higher in infected compared to uninfected peas, thus indicating that the Rhizobium effect allows for favorable management of trade-offs between nodules growth for nitrogen capture and root foraging for water and other nutrient uptake in pea. The outcome of this basic research can be used to produce molecular tools for breeding pea genotypes able to develop deep-foraging and branched root systems, and more competitive architectures and molecular levels for soil NO3- absorption during seedling establishment without jeopardizing nodulation.

Quantitative trait loci analysis reveals a correlation between the ratio of sucrose/raffinose family oligosaccharides and seed vigour in Medicago truncatula.

Seed vigour is important for successful establishment and high yield, especially under suboptimal environmental conditions. In legumes, raffinose oligosaccharide family (RFO) sugars have been proposed as an easily available energy reserve for seedling establishment. In this study, we investigated whether the composition or amount of soluble sugars (sucrose and RFO) is part of the genetic determinants of seed vigour of Medicago truncatula using two recombinant inbred line (RIL) populations. Quantitative trait loci (QTL) mapping for germination rate, hypocotyl and radicle growth under water deficit and nutritional stress, seed weight and soluble sugar content was performed using RIL populations LR1 and LR4. Seven of the 12 chromosomal regions containing QTL for germination rate or post-germinative radicle growth under optimal or stress conditions co-located with Suc/RFO QTL. A significant negative correlation was also found between seed vigour traits and Suc/RFO. In addition, one QTL that explained 80% of the variation in the ratio stachyose/verbascose co-located with a stachyose synthase gene whose expression profile in the parental lines could explain the variation in oligosaccharide composition. The correlation and co-location of Suc/RFO ratio with germination and radicle growth QTL suggest that an increased Suc/RFO ratio in seeds of M. truncatula might negatively affect seed vigour.

Characterization of a dual-affinity nitrate transporter MtNRT1.3 in the model legume Medicago truncatula.

Primary root growth in the absence or presence of exogenous NO(3)(-) was studied by a quantitative genetic approach in a recombinant inbred line (RIL) population of Medicago truncatula. A quantitative trait locus (QTL) on chromosome 5 appeared to be particularly relevant because it was seen in both N-free medium (LOD score 5.7; R(2)=13.7) and medium supplied with NO(3)(-) (LOD score, 9.5; R(2)=21.1) which indicates that it would be independent of the general nutritional status. Due to its localization exactly at the peak of this QTL, the putative NRT1-NO(3)(-) transporter (Medtr5g093170.1), closely related to Arabidopsis AtNRT1.3, a putative low-affinity nitrate transporter, appeared to be a significant candidate involved in the control of primary root growth and NO(3)(-) sensing. Functional characterization in Xenopus oocytes using both electrophysiological and (15)NO(3)(-) uptake approaches showed that Medtr5g093170.1, named MtNRT1.3, encodes a dual-affinity NO(3)(-) transporter similar to the AtNRT1.1 'transceptor' in Arabidopsis. MtNRT1.3 expression is developmentally regulated in roots, with increasing expression after completion of germination in N-free medium. In contrast to members of the NRT1 superfamily characterized so far, MtNRT1.3 is environmentally up-regulated by the absence of NO(3)(-) and down-regulated by the addition of the ion to the roots. Split-root experiments showed that the increased expression stimulated by the absence of NO(3)(-) was not the result of a systemic signalling of plant N status. The results suggest that MtNRT1.3 is involved in the response to N limitation, which increases the ability of the plant to acquire NO(3)(-) under N-limiting conditions.

Nitrate inhibits primary root growth by reducing accumulation of reactive oxygen species in the root tip in Medicago truncatula.

In Medicago truncatula, nitrate, acting as a signal perceived by NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER FAMILY 6.8 (MtNPF6.8), inhibits primary root growth through a reduction of root cell elongation. Since reactive oxygen species (ROS) produced and converted in root tip (O2•- → H2O2 → •OH) have been reported to control cell elongation, the impact of nitrate on the distribution of these ROS in the primary root of M. truncatula was analyzed. We found that nitrate reduced the content of O2•-, H2O2 and •OH in the root tip of three wild type genotypes sensitive to nitrate (R108, DZA, A17), inhibition of root growth and O2•- accumulation being highly correlated. Nitrate also modified the capacity of R108 root tip to produce or remove ROS. The ROS content decrease observed in R108 in response to nitrate is linked to changes in peroxidase activity (EC1.11.1.7) with an increase in peroxidative activity that scavenge H2O2 and a decrease in hydroxylic activity that converts H2O2 into •OH. These changes impair the accumulation of H2O2 and then the accumulation of •OH, the species responsible for cell wall loosening and cell elongation. Accordingly, nitrate inhibitory effect was abolished by externally added H2O2 or mimicked by KI, an H2O2 scavenger. In contrast, nitrate has no effect on ROS production or removal capacities in npf6.8-2, a knockdown line insensitive to nitrate, affected in the nitrate transporter MtNPF6.8 (in R108 background) by RNAi. Altogether, our data show that ROS are mediators acting downstream of MtNPF6.8 in the nitrate signaling pathway.

Involvement of Medicago truncatula glutamate receptor-like channels in nitric oxide production under short-term water deficit stress.

Early stages of plant development are highly susceptible to environmental cues, and seedlings have to develop sophisticated mechanisms to sense and respond to abiotic stresses. We have previously identified that abscisic acid (ABA), nitric oxide (NO) and modulation of nitrogen metabolism are involved in adaptive responses in Medicago truncatula seedlings under water deficit stress. Here, we investigated whether glutamate receptor-like channels (GLRs) played a role in the developmental physiological processes of Medicago seedlings during post-germination after a short-term water deficit stress. Twenty-nine independent MtGLR genes have been identified and then divided into four clades following a phylogenetic analysis; seventeen of them exhibited specific domains which are characteristic of animal ionotropic glutamate receptors. Under drought stress, ABA-induced NO accumulation was significantly reduced in presence of a GLR competitive antagonist, suggesting that this water deficit-induced endogenous NO production was mediated through a MtGLR-dependent pathway. Water deficit-induced inhibition of embryo axis elongation was strongly reduced whereas loss of water content was alleviated when MtGLRs were inhibited. These results suggest that glutamate receptors-like channels are required, through their involvement in NO production, in adaptive responses under short-term water-deficit stress during Medicago seedling establishment.

Differential transcription initiation and alternative RNA splicing of Knox7, a class 2 homeobox gene of maize.

Knox7, a class 2 homeobox gene has been characterized in maize. A combination of experimental (3'- and 5'-RACE) and bioinformatics approaches supported the idea that Knox7 would be transcribed into two alternative transcripts by differential initiation of transcription. Sequence differences between alternative transcripts, Knox7L the larger and Knox7S the smaller, were confined to their 5' end regions and exon 1 was only found in Knox7L transcripts. Deduced proteins shared the same homeodomain, while an Ala and Ala/Gly rich domain was found only in KNOX7L protein. We hypothesize that KNOX7L and KNOX7S might regulate (differentially) the expression of the same gene(s) by binding competitively to the same cis-acting element(s). Further expression analysis using RT-PCR to amplify cDNA portions corresponding to ORFs of both Knox7 alternative transcripts showed that seven cDNA clones were probably generated by alternative splicing of Knox7L. Alignment of these sequences showed that they are in frame suggesting the existence of the corresponding proteins. Quantitative RT-PCR experiments indicated that Knox7S and Knox7L were expressed in maize embryos during germination. In the same tissue, expression of Knox7S was stimulated by light and ABA and inhibited by GA, two hormones that control germination process.

Physiological and molecular aspects of aspartate-derived amino acid metabolism during germination and post-germination growth in two maize genotypes differing in germination efficiency.

The Asp-derived amino acid pathway has been studied during the early stages of development in two maize genotypes, Io and F2, differing in germination efficiency and post-germination growth. In both genotypes expression of Ask2 (monofunctional Asp-kinase-2), Akh1 and Akh2 (bifunctional Asp-kinase-homo-Ser dehydrogenase-1 and 2), increased throughout germination and post-germination growth, suggesting a developmental regulation, whereas Ask1 (monofunctional Asp-kinase-1) was expressed constitutively. The major difference between Io and F2 concerned genes encoding bifunctional enzymes, particularly Akh2, the expression of which was dramatically low in F2. 15N-Asp labelling showed differences in in vivo Asp-kinase activities between the genotypes studied. Asp flux through the Met/Thr branches was higher in Io than in F2, while the latter exhibited a higher flux of Asp through the Lys branch. Physiological results, together with the higher Akh2 expression in Io, suggest that bifunctional enzyme activity, favourable to Met/Thr, was higher in Io than in F2 and that the monofunctional pathway was boosted in F2 because of the lower competition by the bifunctional pathway, thus allowing for higher flux of Asp through the Lys branch. In conclusion, it is suggested that F2 germination and post-germination growth might have been partially inhibited due to a limitation in Met and Thr availability. A negative physiological effect related to Lys accumulation in F2 is also discussed.

A gene encoding a germin-like protein, identified by a cDNA-AFLP approach, is specifically expressed during germination of Phaseolus vulgaris.

In order to identify markers of germination in Phaseolus vulgaris L., a cDNA-amplified fragment length polymorphism (AFLP) approach was conducted on mRNAs from embryo axes and from cotyledons. Among changes observed throughout the germination process, a cDNA fragment not detected 9 h after imbibition (HAI) but present specifically in axes 24 HAI was further studied. The complete cDNA was recovered by rapid amplification of cDNA ends, then cloned and sequenced. It includes an open reading frame predicting a 206-amino-acid polypeptide of 21.8 kDa. Analysis of the nucleotide sequence and deduced amino acid sequence revealed a high homology with germin-like proteins (GLPs), and particularly with an auxin-binding protein from peach, ABP19, that belongs to the GLP family. Thus, we propose that this cDNA encodes the first GLP described in P. vulgaris, designated PvGLP1. Northern blot analysis carried out on mRNAs from seed axes showed a dramatic increase in PvGLP1 expression a few hours before radicle emergence (17 HAI). Among mature vegetative tissues, PvGLP1 expression was very weak in pods and not detected in leaves, stems or roots. Immunoblot analysis using antibodies raised against AtGER3 from Arabidopsis thaliana showed that the protein could be detected only in axes from the dry seed stage onwards, at a steady-state level. Then, PvGLP1 expression seems to be associated with the early stages of embryo axis growth. The high homology indicated with ABP19 led us to study the effect of different concentrations of indole-3-acetic acid (IAA) on PvGLP1 expression during germination. Whereas no effect was noticed at low concentrations (1, 5, 10 microM), a marked decrease in PvGLP1 mRNA level was observed in axes of seeds imbibed with 100 microM IAA. Thus, PvGLP1 gene expression is not stimulated by auxin and, moreover, it might be inhibited by high concentrations of IAA.